Protective Effects of Anethole in Foeniculum vulgare Mill. Seed Ethanol Extract on Hypoxia/Reoxygenation Injury in H9C2 Heart Myoblast Cells

Abstract

Background: Active compounds from plants and herbs are increasingly incorporated into modern medical systems to address cardiovascular diseases (CVDs). Foeniculum vulgare Mill., commonly known as fennel, is an aromatic medicinal plant and culinary herb that is popular worldwide.

Methods: Protective effects against cellular damage were assessed in the H9C2 cardiomyocyte hypoxia/reoxygenation (H/R) experimental model. The identities of phytochemicals in FVSE were determined by GC-MS analysis. The phytochemical's potential for nutrients and pharmacokinetic properties was assessed by ADMET analysis.

Results: GC-MS analysis of the ethanol extracts of F. vulgare identified 41 bioactive compounds, with four prominent ones: anethole, 1-(4-methoxyphenyl)-2-propanone, ethoxydimethylphenylsilane, and para-anisaldehyde diethyl acetal. Among these, anethole stands out due to its potential for nutrients and pharmacokinetic properties assessed by ADMET analysis, such as bioavailability, lipophilicity, flexibility, and compliance with Lipinski's Rule of Five. In the H/R injury model of H9C2 heart myoblast cells, FVSE and anethole suppressed H/R-induced reactive oxygen species (ROS) generation, DNA double-strand break damage, nuclear condensation, and the dissipation of mitochondrial membrane potential (ΔΨm).

Conclusions: These findings highlight the therapeutic potential of FVSE and its prominent component, anethole, in the treatment of CVDs, particularly those associated with hypoxia-induced damage.

Keywords: ADMET; DNA double-strand break damage; Foeniculum vulgare Mill.; GC-MS; H9C2; ROS; boiled-egg model; mitochondrial membrane potential; nuclear condensation.

Figure 1

The GC-MS profile of FVSE. The prominent peaks are identified as anethole (a), 1-(4-methoxyphenyl)-2-propanone (b), ethoxydimethylphenylsilane (c), and para-anisaldehyde diethyl acetal (d).

Figure 2

Identities of the four prominent components in Foeniculum vulgare Mill. seed ethanol extract (FVSE). (A) MS fragmentation patterns. (B) Molecular structures. (a) Anethole, (b) 1-(4-methoxyphenyl)-2-propanone, (c) ethoxydimethylphenyl-silane, and (d) para-anisaldehyde diethyl acetal. See Supplementary Figure S2 for more extensive data.

Figure 3

Pharmacokinetic properties generated with the SwissADME web tool. (A) A boiled-egg model. Wildman–Crippen LogP (WLOGP). Topological polar surface area (TPSA). (B) Radar plots. (a) Anethole, (b) 1-(4-methoxyphenyl)-2-propanone, (c) ethoxydimethylphenyl-silane, and (d) para-anisaldehyde diethyl acetal. Lipophilicity (LIPO), SIZE (MW, g/mol), polarity (POLAR), insolubility (INSOLU), instauration (INSATU), and flexibility (FLEX).

Figure 4

Effects of FVSE and anethole on the viability of H9C2 cells under H/R treatment. (A,B) Representative images showing a marked increase in cell numbers in H/R treatment. Scale bar: 50 µm. (Ca,Da) The numbers of all attached cells. (Cb,Db) The proportions of viable cells among attached cells, assessed by trypan blue exclusion assays. All statistics are expressed as means ± SEM. # number, * p < 0.05, *** p < 0.001 (ANOVA).

Figure 5

FVSE and anethole suppress ROS Production. Cells were grown and H/R-treated, as described in Figure 4, and stained with DCFDA. (A) Representative images showing ROS-producing cells. Scale bar: 50 µm. (B) Measurement of ROS-positive cells (a) and the relative intensity (arbitrary units, a.u.) (b). Bars represent the mean ± SEM (n = 3, ~200–300 cells per group). *** p < 0.001 (ANOVA).

Figure 6

FVSE and anethole mitigate double-strand DNA breakage in H/R. Cells were grown and subjected to H/R shock, as described in Figure 4, then immunostained with an anti-phospho-H2AX antibody and stained with DAPI to reveal nuclei. (A) Representative images showing phospho-H2AX puncta. Scale bar: 10 µm. (B) Statistical analysis. Bars represent means ± SEM (n = 30 nuclei). *** p < 0.001 (ANOVA).

Figure 7

FVSE and anethole prevent nuclear condensation in H/R. H9C2 cells were grown and subjected to H/R shock, as described in Figure 4, then stained with DAPI to reveal nuclei. The condensed nuclei are marked by yellow arrowheads. (A) Representative images showing cells with condensed nuclei. Scale bar: 50 µm. (B) Statistical analysis. Bars represent means ± SEM (n = ~200–300 nuclei, 3 replicates). *** p < 0.001 (ANOVA).

Figure 8

FVSE and anethole prevent ΔΨm dissipation in H/R. H9C2 cells were grown and subjected to H/R shock, as described in Figure 4, and ΔΨm was determined by JC-1 staining. (A) Representative images showing red and green fluorescence. Scale bar: 50 μm. (B) Statistical analysis. Bars represent means ± SEM (n = ~200–300 spots). ** p < 0.01, *** p < 0.001 (ANOVA).